During this kpast year we have attempted to develop systematic methods by which we could study the roles of modified nucleotides in mammalian tRNAs by cloning tRNA genes into various stable RNA prompter containing plasmids. In these experiments we have sub-cloned two fragments of the human mitochondrial genome (as a model system which encodes for several tRNAs in either orientation) into a modified pKG-1800 plasmid (pLG-110). This chimeric plasmid has the tRNA genes inserted between an E. Coil leucine tRNA promoter and the galactokinase gene. As these chimeric plasmids express the galactokinase activity in vivo, they may be ideal candidates for use either in vivo and/or in vitor expression vectors. We have recently isolated mg quantities of several coloned tRNA genes and presently are studying their transcription in vitro. Once we obtained sufficient quantities of well characterized in vitro transcription products, we will then proceed to study in vitor RNA processing. Furthermore, we will attempt to create a series of "point altered" tRNAs which can then be used in additional studies aimed at determining the effect of several specific modified nucleotides on tRNA functions.